Project 1: Wegener's granulomatosis (WG) is a granulomatous vasculitis that affects the upper respiratory tract, lung and kidney. In the present study we investigated the capacity of circulating T cells and macrophages to produce cytokines, since these cell types make up a significant proportion of cells infiltrating the granulomatous lesions. In studies of proliferate responses of circulating T cells, we showed that T cells from active WG patients (n=11) exhibited greatly enhanced PMA/lonomycin- or anti-CD3/anti-CD28-induced proliferation compared with either inactive patients (n=6) or normal controls (n=12). Interestingly, this difference disappeared if DR+ T cells were removed from the cell populations studied, suggesting that the abnormality was limited to previously activated cells that had taken part in the disease process. In studies of cytokine secretion of circulating cells, we found that T cells from active WG patients secreted greatly increased amounts of IFN-gamma compared to inactive WG patients or normal controls, particularly when purified CD4+/DR+ T cells were studied. However, no differences were observed in the secretion of IL-4, IL-5 or IL-10. In further studies of cytokine secretion in WG, we showed that whereas monocytes from active WG patients produced normal amounts of TNF-alpha compared to normal controls, T cells from this group produced significantly elevated amounts of TNF-alpha. Taken together, these studies indicate that active WG is associated with a Th1 T cell cytokine secretion profile. Finally, to determine the mechanism of the Th1 T cell abnormality, we investigated WG patients for their capacity to secrete IL-12, the cytokine that drives Th1 T cell differentiation. In these studies we showed that LPS- or CD40L-stimulated monocytes from WG patients produced increased amounts of IL-12. In addition, we showed that IFN-gamma production by WG T cells is inhibited by IL-10, a cytokine that down-regulates IFN-gamma production by its negative effects on IL-12 production. These data suggest that the overproduction of Th1 cytokines such as IFN-gamma and TNF-alpha, secondary to dysregulated IL-12 secretion, plays an important role in the pathogenesis of WG. In addition, they indicate that IL-10 administration could be a useful therapy in this disease. Project 2: Intestinal lymphangiectasia (IL) is a disease of the lymphatics marked by blocked, dilated intestinal lymphatic vessels and the loss of lymphatic fluid into the gastrointestinal tract. Previous studies have revealed that that loss process in IL is characterized, not only by lymphocytopenia, but also by depletion of lymphocytes in lymphoid tissues and diminished delayed skin test reactivity. This latter in vivo evidence of diminished T cell function was initially attributed to a quantitative T cell defect, however, it was subsequently learned that patient T cells manifest poor in vitro proliferative responses to antigens and mitogens even when T cell numbers are normalized in vitro cultures. This implies that a qualitative as well as a quantitative disorder of T cells is present in IL, one perhaps due to preferential loss of responsive lymphocyte subpopulations. In the present study we re-examined the question of selective lymphocyte loss in IL utilizing recently available monoclonal antibodies to identify and isolate lymphocyte subsets. We found that while all T cell subsets were subject to intestinal loss in IL, there is selective loss of CD4+/CD45RA+ T cells so that the patient circulating T cell population contains very few cells of this subset. In addition, the residual CD4+CD45RO+ T cells are over-represented by previously sensitized DR+ T cells and by CD45RO+ T cells which lack the CD27+ marker. Finally, the circulating cells are enriched in T cells which proliferate poorly and which produce increased amounts of IL-4 and decreased amounts of IFN-gamma. These results indicate that the cells at risk for loss in IL are naive T cells which are circulating through the lymphatics rather than mature cells in the lamina propria. In addition, they indicate that the mature cells in the circulation that survive the loss process are skewed toward the Th2 T cell phenotype. Project 3: Whipple's disease (WD) is a systemic infection with the recently identified actinomycete, T. whippelii. Disease is most evident in the GI tract, where one sees the characteristic accumulation of PAS-positive of bacteria and bacterial remnants in macrophages. To determine if this infection is associated with a host immune defect, we tested highly purified monocytes and peripheral blood mononuclear cells (PBMC) from two WD patients and two relatives for the in vitro production of interleukin-12 (IL-12), IL-10, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and transforming growth factor-beta (TGF-beta), in addition, we measured serum levels of these cytokines and determined cytokine expression in duodenal biopsies from three patients. We found a highly reduced monocyte IL-12 production and a decreased IFN-gamma secretion by PBMC in vitro, as well as reduced immunohistological staining for IL-12 and IFN-gamma, but no decrease in other cytokines in WD patients. A similar but less severe defect in two WD relatives argued for a genetic basis of this abnormality. Serum IgG2, a IFN-gamma dependent Ig subclass, and serum TGF-beta levels were reduced in WD patients. These results indicate that monocyte defects in WD which may result in secondary reduction of IFN-gamma production and decreased IgG2 serum levels. These results thus proved a rationale for additive immunotherapy in patients with antibiotic-refractory WD.